User:Brendan J. Hussey/Notebook/Pharmbiants

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Project Description/Abstract

Project Pharmbiants was created with the intention of utilizing the symbiotic relationship that mammals share with their commensal microbes as a means for delivering biological compounds to the host. Particularly, genetic engineering/synthetic biology approaches will be used to modify commensal gut microorganisms to deliver therapeutically important compounds. Project Pharmbiants is partially an attempt at addressing the current limitations of gene therapy (that is the lack of safe, reliable, site specific recombination) for the treatment of disease in mammals. Microorganisms are much easier, safer and more predictable to engineer then eukaryotic systems and could potentially be used to monitor and deliver gene products to therapeutic affect. Additionally, this technology could be used as an extension of probiotics to deliver any number of compounds to promote health and well being. This project has a number of proposed stages, the development of which depend on available facilities and time (as this is currently a "weekend project", although i would be excited with any professional opportunity).

  • Phase 1. Produce a number of small molecule compounds in commensal bacteria and deliver them to the host through production in fermented food products. The bacteria, such as those from the Lacctocossus and Lactobaccilus genus, will grow and produce compounds in common fermentation setups, such as those used for making yogurt, and will be deactivated before consumption. Optimization of genetic networks to maximize yields will be the primary concern at this stage as well as experimentation with the type of compounds which can be successfully produced and made bioavailable, mostly small molecules.


  • Phase 2. Design commensal gut microbes that sustain a stable population in the gut and deliver a wide range of compounds to the host. This phase will attempt to expand the number of compounds that can be produced and made bioavailable to include small peptides upto large multimeric protein compounds. It will also involve the delivery of live bacteria into the large intestine through consumables. Integration and coupling of population regulation circuits (quorum sensing), protein glycosylation and excretion systems as well as host metabolite monitoring will be considered and tested in vitro.

Phase 1 Details

Target species

L.reuteri

  • Gram positive
  • Common non pathogenic, gut commensal
  • High viability in yogurt culture as well as gut
  • Lacks histidine metabolism

Compound(s) of interest

AICAR (5'-phosphoribosyl-4-carboxamide-5-aminoimidazole)

  • Orally active exercise mimetic
  • Possible oral treatment for muscle wasting diseases

Possible Metabolic Manipulations

Histine operon introduction

  • AICAR as byproduct
  • Very large operon
  • Energetically expensive (≈41 ATP per histine/AICAR)

Purine metabolism modification (current choice)

  • AICAR as purine intermediate in IMP biosynthesis
  • Purine metabolism essential for life in common media
  • Extensive modification required

Prosed Protocol

Identify Desired Purine Metabolic Alterations (completed)

  • LAR 0138 (IMP cyclohydrolase; last step in IMP synthesis)
    • catalyzes both the conversion of AICAR to FAICAR and from FAICAR to IMP
    • to knock out via loxp/cre when bacterial population plateaus in culure
    • knock out in humans by natural mutation causes massive AICAR increase
  • LAR 0135 (purF)
    • rate limiting step in IMP synthesis
    • converts PRPP/L-glutamine into ribosylamine-5P
    • feedback resistant strains have high IMP yeilds (espeically when combined with prs)
  • LAR 0212 (prs)
    • converts Ribose-5P into PRPP (precedes purF)
    • feedback resistant strains have high IMP yields (espeically when combined with purF)

Vector Selection and Design

  • pNZ5319
    • double recombination vector for Lactobacilli sp.
    • Add LAR 0138 before P32 CAT, behind strong constitutive promoter
  • pRV566
    • Protein expression vector for Lactobacilli sp.
    • To constitutively express feedback resistant LAR 0135 and LAR 0212
    • To contain inducible Cre

Digital Reconstruction

  • pNZ5319 (construction in progress)
    • no digital construction available
    • modified from pGIZ850
  • pGIZ850 (construction complete)
    • no digital construction available
    • modified from pUC18Ery

Notes

  • This project is now being considered for redirection as part of potential thesis work. As of now, this particular project is suspended.
  • This project was initiated in hardcopy April 30th 2009 (in a lab notebook) and in the works as early as beginning of 2008. This online Notebook was started March 21st, 2010. I plan to type out all the pages of my lab notebook and provide figures/documents/data unedited to show my thought process.
  • Your comments/suggestions are greatly appreciated.

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